Extended Data Figure 4.

a, The atp-2(RNAi) worms have higher levels of DCF fluorescence than gfp control worms (P < 0.0001, by t-test, two-tailed, two-sample unequal variance). Supplementation with α-KG also leads to higher DCF fluorescence, in both HT115 (for RNAi) and OP50 fed worms (P = 0.0007, and P = 0.0012, respectively). ROS levels were measured using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCF-DA). Since whole worm lysates were used, total cellular oxidative stress was measured here. H₂DCF-DA (Molecular Probes, D399) was dissolved in ethanol to a stock concentration of 1.5 mg/mL. Fresh stock was prepared every time prior to use. For measuring ROS in worm lysates, a working concentration of H₂DCF-DA at 30 ng/mL was hydrolyzed by 0.1 M NaOH at room temperature for 30 min to generate 2’, 7’-dichlorodihydrofluorescein (DCFH) before mixing with whole worm lysates in a black 96-well plate (Greiner Bio-One). Oxidation of DCFH by ROS yields the highly fluorescent 2', 7'-dichlorofluorescein (DCF). DCF fluorescence was read at excitation / emission of 485 / 530 nm using SpectraMax MS (Molecular Devices). H₂O₂ was used as positive control (not shown). To prepare the worm lysates, synchronized young adult animals were cultivated on plates containing vehicle or 8 mM α-KG and OP50 or HT115 E. coli for 1 day, and then collected and lysed as described in “Assay for ATP levels in C. elegans” (see Methods). Mean ± s.d. is plotted. b, There was no significant change in protein oxidation upon α-KG treatment or atp-2(RNAi). Oxidized protein levels were determined by the OxyBlot. Synchronized young adult N2 animals were placed onto plates containing vehicle or 8 mM α-KG, and seeded with OP50 or HT115 bacteria that expressed control or atp-2 dsRNA. Adult day 2 and day 3 worms were collected and washed 4 times with M9 buffer, and then stored at −80 °C for at least 24 h. Laemmli buffer (Biorad, 161-0737) was added to every sample and animals were lysed by alternate boil/freeze cycles. Lysed animals were centrifuged at 14,000 rpm for 10 min at 4 °C to pellet worm debris, and supernatant was collected for oxyblot analysis. Protein concentration of samples was determined by the 660 nm Protein Assay (Thermo Scientific, 1861426) and normalized for all samples. Carbonylation of proteins in each sample was detected using the OxyBlot Protein Oxidation Detection Kit (Millipore, S7150).